Stable, high relaxivity MRI contrast agents Abstract Contrast agents play an important role in clinical magnetic resonance imaging (MRI). About one third of scans employ a contrast agent and these agents are almost exclusively gadolinium (Gd) based T1 agents that provide positive image contrast. However, Gd-based agents have been implicated in nephrogenic systemic fibrosis (NSF), a debilitating and life threatening disorder. It is hypothesized that Gd release from the contrast agent is a causative factor. New contrast agents, either extracellular tracers or targeted molecular imaging agents, must address the risk of Gd toxicity. Two additional features are required to make this new contrast agent technology broadly applicable today and in the future. First, although the majority of clinical scans today ar performed at 1.5 Tesla, there is a steady increase in imaging performed at higher fields. T1 relaxivity (positive contrast) typically decreases with increasing field while T2 relaxation (signa loss) becomes more efficient. Successful strategies for increasing r1 at low fields can be detrimental at high fields. New agents should demonstrate improved relaxivity from 1.5T to 7T and higher fields relative to existing contrast media. Second, new applications in molecular imaging require bifunctional molecules: a targeting group like a peptide linked to contrast agent group. New contrast agents should be easily incorporated into molecular imaging probes without loss of their relaxometric properties. We have developed a platform technology based on the amino acid chelator DOTAla that addresses this problem in two ways. The DOTAla moiety is a macrocyclic chelator that forms Gd complexes that are extremely inert to decomplexation and transmetallation, and is comparable to the macrocyclic contrast agents used clinically in this regard. The Gd-DOTAla complex can be incorporated into peptides in a rigid fashion in order to generate contrast agents that have high relaxivity as a result of optimized correlation times and increased gadolinium content per molecule. Because DOTAla is an amino acid, the synthetic flexibility in creating multimeric, targeted, and/or multimodal probes is unlimited. Here we build on this technology platform to develop optimized bifunctional reagents that deliver enhanced relaxivity at high fields. In Aim 1 we take three complimentary approaches to synthesize compounds with enhanced relaxivity. In Aim 2 we will perform extensive biophysical characterization of the compounds prepared in Aim 1 using advanced magnetic resonance and computational techniques in order to create models that prospectively predict new compounds with optimized dynamics and relaxivity. We then demonstrate the broader utility of this optimized technology platform by preparing untargeted (Aim 3) and peptide-targeted (Aim 4) high relaxivity contrast agents, and characterize them in rodent models of brain cancer and stroke.